Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
  • A61K48/0091—Purification or manufacturing processes for gene therapy compositions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/02—Inorganic compounds
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
  • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
  • A61K47/26—Carbohydrates, e.g. sugar alcohols, amino sugars, nucleic acids, mono-, di- or oligo-saccharides; Derivatives thereof, e.g. polysorbates, sorbitan fatty acid esters or glycyrrhizin
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/0012—Galenical forms characterised by the site of application
  • A61K9/0019—Injectable compositions; Intramuscular, intravenous, arterial, subcutaneous administration; Compositions to be administered through the skin in an invasive manner
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/08—Solutions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K9/00—Medicinal preparations characterised by special physical form
  • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
  • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/04—Antibacterial agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/10—Antimycotics
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
  • A61P31/12—Antivirals
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P33/00—Antiparasitic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P37/00—Drugs for immunological or allergic disorders
  • A61P37/02—Immunomodulators
  • A61P37/04—Immunostimulants
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
  • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
  • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
  • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
  • C12N15/86—Viral vectors
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10111—Atadenovirus, e.g. ovine adenovirus D
  • C12N2710/10132—Use of virus as therapeutic agent, other than vaccine, e.g. as cytolytic agent
• • C—CHEMISTRY; METALLURGY
  • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
  • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
  • C12N2710/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA dsDNA viruses
  • C12N2710/00011—Details
  • C12N2710/10011—Adenoviridae
  • C12N2710/10111—Atadenovirus, e.g. ovine adenovirus D
  • C12N2710/10141—Use of virus, viral particle or viral elements as a vector
  • C12N2710/10143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Definitions

• the present invention relates to the formulation of adenoviral vectors in freeze dried compositions, their formulations as well as methods for obtaining the freeze-dried composition.
• Adenoviral vectors represent a prophylactic or therapeutic protein delivery platform whereby the nucleic acid sequence encoding the prophylactic or therapeutic protein is incorporated into the adenoviral genome, which is brought to expression when the adenoviral particle is administered to the treated subject. It has been a challenge in the art to develop stabilizing formulations for the adenoviral vectors which allow storage at acceptable storage temperatures with a considerable shelf life. Stabilizing formulations have been reported for human adenoviral vectors such as described by R.K Evans et al. ('Development of stable Liquid Formulations for Adenovirus-Based Vaccines' Journal of Pharmaceutical Sciences (2004) Vol. 93, No. 10, 2458-2475). However, there remains a need in the art for formulations preserving the stability of adenoviral vectors.
• the inventors surprisingly found that the use of sorbitol in the formulation of simian adenoviral vectors substantially improves stability throughout lyophilisation, especially in combination with the amorphous sugar trehalose as a further cryoprotectant.
• the invention therefore provides an aqueous mixture for lyophilisation and a freeze-dried composition obtained from said aqueous mixture by lyophilisation (hereinafter referred to as the "dried composition") comprising sorbitol in combination with a further amorphous sugar acting as a cryoprotectant, such as trehalose.
• the invention further provides the adenoviral compositions containing sorbitol and a further amorphous sugar also comprising a low amount of NaCI.
• the invention also provides a method of using the freeze-dried composition whereby the composition is reconstituted with a low salt aqueous liquid, e.g. water for injection or an aqueous solution of a non- ionic isotonifying agent.
• the invention further provides a method of lyophilisation of the described adenoviral vector compositions.
• FIG. 1 illustration of the freeze drying cycle as used in Example 1.
• Figure 3 infectivity of the adenoparticles contained in the compositions as obtained in Example 1: (1) trehalose 23% (designated by (+), (2) sucrose 23% (designated by X), (3) trehalose 23% + sorbitol 2% (designated by (Y), (4) sucrose 23% + sorbitol 2% (designated by (Z) and (5) Fresh purified bulk control (designated by upright triangle) and (6) degraded purified bulk control (designated by inverted triangle).
• Figure 4 illustration of freeze-drying cycle as used in Example 2.
• Figure 5 Tg as determined in Example 2.
• Figure 6 moisture content as determined in Example 2.
• Figure 7 - osmolality as determined in Example 2.
• Figure 8 PicoGreen data as determined in Example 2.
• Figure 9 infectivity as determined in Example 2.
• Figure 11 illustration of the statistical analysis of data with respect to different parameters as determined in Example 2 to identify optimized combinations (design of experiments (DOE) plot 1).
• Figure 12 illustration of the statistical analysis of data with respect to different parameters as determined in Example 2 to identify optimized combinations wherein higher trehalose concentrations are allowed (DOE plot 2).
• Figure 13 freeze drying cycle 1 as used in Example 3.
• Figure 14 freeze drying cycle 2 as used in Example 3.
• Figure 15 PicoGreen data as determined in Example 3: (x) data points for samples obtained using freeze-drying condition with secondary desiccation at +10°C (lyo cycle (2)), (+) data points for samples obtained using freeze-drying condition with secondary desiccation at +25°C (lyo cycle (1)), (upright triangle)- control adenoviral stock, (inverted triangle) - negative control degraded adenoviral stock.
• Figure 16 - infectivity data as determined in Example 3 (x) data points for samples obtained using a freeze-drying condition with secondary desiccation at +10°C, (+) data points for samples obtained using freeze-drying condition with secondary desiccation at +25°C, (upright )triangle - control adenoviral stock, (inverted triangle) - negative control degraded adenoviral stock.
• Figure 17 - U PLC data as determined in Example 3 (x) data points for samples obtained using freeze- drying condition with secondary desiccation at +10°C, (+) data points for samples obtained using freeze-drying condition with secondary desiccation at +25°C, (upright triangle)- control adenoviral stock, (inverted triangle) - negative control degraded adenoviral stock.
• Figure 19 scatter plot demonstrating the stability of trehalose 18% + sorbitol 3.5% and trehalose 23% at drying temperatures of 15°C and 25°C for 200 days.
• cryoprotectant refers to a class of excipients which prevents freeze damage of what is being frozen, in casu, the adenoviral vector.
• An amorphous sugar suitable for use according to the present invention in combination with sorbitol may be selected from sucrose, trehalose, mannose, mannitol, raffinose, lactitol, lactobionic acid, glucose, maltulose, iso-maltulose, lactulose, maltose, lactose, isomaltose, maltitol, palatinit, stachyose, melezitose, dextran, or a combination thereof.
• the amorphous sugar is selected from sucrose, trehalose, lactose, raffinose, dextran and combinations thereof.
• the further amorphous sugar in combination with sorbitol is trehalose or sucrose, or, trehalose in combination with a second amorphous sugar such as selected from sucrose, lactose, raffinose, dextran and mannitol.
• the amorphous sugar is trehalose, sucrose or combination of sucrose and trehalose.
• the amorphous sugar is trehalose or trehalose in combination with sucrose.
• the amorphous sugar is trehalose.
• Sorbitol and the selected amorphous sugar, e.g. trehalose may be present in a defined ratio.
• the ratio of sorbitol to amorphous sugar is 4/10 or below, 4/12 or below, 4/13 or below, 4/14 or below.
• the ratio of sorbitol to amorphous sugar is between 4/10 and 3/23, between 4/12 and 4/23, between, 4/13 and 4/20, between 4/14 and 4/18, between 4/14 and 3.5/16, or between 4/14 and 4/16.
• the ratio is between 4/14 and 4/16.
• the amorphous sugar is trehalose and the ratio sorbitol to trehalose is between 4/14 and 4/16.
• Sorbitol may be present in defined amounts in the aqueous mixture from which the composition is freeze-dried.
• the aqueous mixture contains between 2 and 4% (w/v), between 2.5 and 4% (w/v) or between 3 and 4% (w/v) of sorbitol.
• sorbitol is present in an amount between 3 and 4% (m/v).
• the amorphous sugar as selected according to the embodiments herein may be present in defined amounts.
• the aqueous mixture contains at least 3% (w/v), at least 5% (w/v), at least 10% (w/v), at least 11% (w/v), at least 12% (w/v), at least 13% (w/v), or at least 14% (w/v) of the amorphous sugar as selected herein above.
• the selected amorphous sugar is present in the aqueous mixture in a total amount of less than 23% (w/v), such as less than 20% (w/v), less than 18% (w/v), less than 17% (w/v), less than 16% (w/v), or less than 15% (w/v).
• the amorphous sugar is present in the aqueous mixture in a total amount of 23% or less (w/v), such as 20% or less (w/v), 18% or less (w/v), 17% or less (w/v), 16% or less (w/v), or 15% or less (w/v).
• the amorphous sugar can be present in the aqueous mixture in a total amount of at least 12%, at least 13% or at least 14% (w/v), but less than 18%, less than 17%, or less than 16% (w/v).
• the amorphous sugar is trehalose and is present in an amount between 12% and 18% (w/v), or, between 14% and 16.5% (w/v).
• adenoviral vectors can be substantially impacted by the presence of salt, such as sodium chloride, either when in dry or when in liquid form.
• the invention thus further relates to formulations, i.e. aqueous mixtures for lyophilisation and dried compositions as described herein, taking into account the sensitivity of adenoviral vectors to salt, such as sodium chloride.
• simian adenoviral vectors are formulated using the aqueous mixtures and dried compositions described herein.
• salt refers to ionic compounds that result from the neutralization reaction of an acid and a base, composed of a related number of cations and anions such that the product is without net charge, for example sodium chloride.
• the component ions can either be inorganic or organic, and, can be monoatomic or polyatomic.
• the amount of salt, in particular the amount of NaCI, present in the aqueous mixture is defined to be less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 15 mM, less than 10 mM, or, less than 7.5 mM.
• the amount of NaCI, present in the aqueous mixture may be defined to be 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 15 mM or less, 10 mM or less, or, 7.5 mM or less.
• the composition is not completely devoid of salt or not completely devoid of NaCI.
• salt in particular sodium chloride
• sodium chloride is present in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM, at least 3 mM, or, at least 4 mM.
• sodium chloride is present in an amount between 1 and 50 mM, between 2.5 and 25 mM, between 2.5 and 15 mM, between 2.5 and 10 mM or between 2.5 and 7.5 mM.
• sodium chloride is present in an amount of about 5 mM, e.g. 5 +/- 0.5 mM.
• the term "between” as used herein is considered to include the end points of the range.
• sodium chloride is said to be present in an amount between 2.5 and 10 mM, those formulations wherein NaCI is present at a concentration of 2.5 mM or 10 mM are included.
• the salt such as sodium chloride
• content of the aqueous liquid or diluent for reconstituting the dried composition is defined.
• reconstituting a freeze-dried composition is meant rehydrating the dried composition to obtain a liquid mixture again.
• the amount of salt e.g.
• sodium chloride, present in the aqueous liquid for reconstituting is less than 50 mM, less than 40 mM, less than 30 mM, less than 20 mM, less than 15 mM, less than 10 mM, or, less than 7.5 mM, 50 mM or less, 40 mM or less, 30 mM or less, 20 mM or less, 15 mM or less, 10 mM or less or 7.5 mM or less.
• the aqueous liquid for reconstituting the lyophilised composition may be essentially free of salt such as essentially free of sodium chloride.
• essentially free is meant that the concentration of salt or sodium chloride is at or very near to zero mM.
• the freeze-dried composition can be reconstituted with water for injection (WFI).
• the aqueous liquid for reconstituting the composition is not completely devoid of salt or sodium chloride.
• salt such as sodium chloride
• salt, such as sodium chloride is present in the aqueous liquid used for reconstituting the composition in an amount between 1 and 50 mM, between 2.5 and 25 mM, between 2.5 and 15 mM, between 2.5 and 10 mM or between 2.5 and 7.5 mM.
• salt such as sodium chloride
• the invention thus also provides a method of using the dried composition as described herein, wherein the dried composition is reconstituted with an aqueous liquid for reconstituting the composition as defined herein.
• the aqueous mixture or dried composition may further include a surfactant selected from poloxamer surfactants (e.g. poloxamer 188), polysorbate surfactants (e.g. polysorbate 80 and/or polysorbate 20), octoxinal surfactants, polidocanol surfactants, polyoxyl stearate surfactants, polyoxyl castor oil surfactants, N-octyl-glucoside surfactants, macrogol 15 hydroxy stearate, and combinations thereof.
• the surfactant is selected from poloxamer surfactants (e.g. poloxamer 188), polysorbate surfactants (e.g. polysorbate 80 and/or polysorbate 20), in particular polysorbate surfactants such as polysorbate 80.
• the surfactant is present in an amount of at least 0.001%, at least 0.005%, at least 0.01% (w/v), and/or up to 0.5% (w/v) as calculated with respect to the aqueous mixture.
• the surfactant can be present in an amount less than 0.25% or less than 0.1% (w/v).
• the surfactant is present in an amount of 0.02% (w/v).
• the surfactant is polysorbate 80 or poloxamer 188 present in the aqueous mixture in an amount between 0.005% and 0.5% (w/v), such as about 0.02% (w/v).
• a buffer is added to the aqueous mixture or dried composition.
• the pH is typically adjusted in view of the therapeutic components of the composition.
• the pH of the aqueous mixture is at least 6, at least 6.5, at least 7 or at least 7.5.
• the pH of the aqueous mixture may be less than 10, less than 9.5, less than 9 or less than 8.5.
• pH of the aqueous mixture is between 6 and 10, between 7 and 9.5, between 7.5 and 9.5, or, about 7.5, for example 7.5 +/-0.5, or, 8.5 +/-0.5.
• the optimal pH is in part also determined by the specific adenoviral vector formulated and/or the transgene incorporated therein.
• An appropriate buffer may be selected from Tris, succinate, borate, Tris-maleate, lysine, histidine, glycine, glycylglycine, citrate, carbonate, phosphate or combinations thereof.
• the buffer is Tris, succinate or borate. In a further embodiment, the buffer is Tris.
• the buffer can be present in the aqueous mixture in an amount of at least 0.5 mM, at least 1 mM, at least 2 mM or at least 5 mM. Or, the buffer can be present in the aqueous mixture in an amount of less than 50 mM, less than 40 mM, less than 30 mM or less than 20 mM. For example, the buffer may be present in an amount of 0.5 mm to 50 mM, 1 mM to 50 mM or 2 mM to 20 mM. In one embodiment, the buffer is present in an amount of about 10 mM. According to specific embodiments, the buffer is Tris, present in the aqueous mixture in an amount between 2 and 20 mM, such as at about 10 mM.
• the composition also comprises histidine in an amount of up to or about 20 mM, such as at a concentration of about 10 mM.
• the composition also comprises bivalent metal ions, such as Mg2+, Ca2+, or Mg2+ or Ca2+ in the form of a salt, such as MgCI2, CaCI2 or MgS04.
• the bivalent metal ion is Mg2+.
• Typical amounts wherein the bivalent metal ions are present in the aqueous mixture are between 0.5 and 10 mM, such as 1 or 2 mM, or 1 mM in particular.
• the specified amounts of excipients considered for inclusion in the composition are typically (and unless otherwise indicated) expressed as w/v% calculated with respect to the volume of the aqueous mixture.
• the amount of excipients may be expressed as w/v% calculated relative to the volume of the reconstituted composition.
• the novel formulation may also increase stability of the adenoviral vector upon storage of the freeze-dried composition.
• the novel formulation allows storage of the composition, liquid or dried, at 4°C, 25°C or 37°C, for up to 1 month, 3 months, 6 months, 1 year, 2 years or 3 years.
• the dried composition can be stored at 4°C for 3 years, at 25°C for 3 months or at 37°C for 1 month. It is understood that storage is adequate if at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the infectivity is retained compared to the infectivity of the starting material.
• the mixtures, compositions and methods described herein allow storage of the adenoviral vector for at least 1 month at 37°C, or at least 3 months at 25°C or at least 3 years at 4°C whilst retaining at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the infectivity compared to the infectivity of the starting material.
• Stability of the adenoviral vectors can, amongst other methods, be determined by measuring the infectivity of the vector, e.g. retention of infectivity upon manipulation (e.g. freeze drying) or storage of the viral vector.
• the term "infectivity” refers to the ability of the vector to enter in a susceptible host, i.e. cells, and deliver its genetic material for expression by the host. Infectivity can be expressed as "the 50% cell culture infectious dose" (CCI D50), which is the amount of adenoviral vector that is required to infect 50% of the cells in a given cell culture. Infectivity can be measured by measuring the proportion of cells wherein an adenoviral transgene is expressed.
• CCI D50 50% cell culture infectious dose
• green fluorescent protein can be used as infectivity marker whereby the number of cells expressing green fluorescent protein after 24 hours of incubation with the vector is determined.
• infectivity can be measured by determination of the number of cells expressing the adenovirus hexon capsid protein after 24 hours of incubation with the vector.
• Adenovirus has been widely used for gene transfer applications due to its ability to achieve highly efficient gene transfer in a variety of target tissues and large transgene capacity.
• Adenoviral vectors of use in the present invention may be derived from a range of mammalian hosts. Over 100 distinct serotypes of adenovirus have been isolated which infect various mammalian species. These adenoviral serotypes have been categorised into six subgenera (A-F; B is subdivided into Bl and B2) according to sequence homology and on their ability to agglutinate red blood cells (Tatsis and Ertl, Molecular Therapy (2004) 10:616-629).
• the adenoviral vector of the present invention is derived from a human adenovirus.
• human-derived adenoviruses are Adl, Ad2, Ad4, Ad5, Ad6, Adll, Ad 24, Ad34, Ad35, particularly Ad5, Adll and Ad35.
• Ad5-based vectors have been used extensively in a number of gene therapy trials, there may be limitations on the use of Ad5 and other human group C adenoviral vectors due to preexisting immunity in the general population due to natural infection. Ad5 and other human group C members tend to be among the most seroprevalent serotypes.
• immunity to existing vectors may develop as a result of exposure to the vector during treatment. These types of preexisting or developed immunity to seroprevalent vectors may limit the effectiveness of gene therapy or vaccination efforts.
• Alternative adenovirus serotypes thus constitute very important targets in the pursuit of gene delivery systems capable of evading the host immune response.
• the adenoviral vector of the present invention is derived from a nonhuman simian adenovirus, also referred to simply as a simian adenovirus.
• a nonhuman simian adenovirus also referred to simply as a simian adenovirus.
• Numerous adenoviruses have been isolated from nonhuman simians such as chimpanzees, bonobos, rhesus macaques and gorillas, and vectors derived from these adenoviruses induce strong immune responses to transgenes encoded by these vectors (Colloca et al. (2012) 5c/. Transl. Med. 4: 1-9; Roy et al. (2004) Virol. 324: 361- 372; Roy et al. (2010) J. Gene Med. 13:17-25).
• vectors based on nonhuman simian adenoviruses include the relative lack of cross-neutralising antibodies to these adenoviruses in the target population. For example, cross-reaction of certain chimpanzee adenoviruses with pre- existing neutralizing antibody responses is only present in 2% of the target population compared with 35% in the case of certain candidate human adenovirus vectors.
• the adenoviral vector is derived from a non-human adenovirus, such as a simian adenovirus and in particular a chimpanzee adenovirus such as ChAd3, ChAd63, ChAd83, ChAdl55, ChAdl57, Pan 5, Pan 6, Pan 7 (also referred to as C7) or Pan 9.
• a non-human adenovirus such as a simian adenovirus and in particular a chimpanzee adenovirus such as ChAd3, ChAd63, ChAd83, ChAdl55, ChAdl57, Pan 5, Pan 6, Pan 7 (also referred to as C7) or Pan 9.
• a chimpanzee adenovirus such as ChAd3, ChAd63, ChAd83, ChAdl55, ChAdl57, Pan 5, Pan 6, Pan 7 (also referred to as C7) or Pan 9.
• Examples of such strains are described in WO03/000
• adenoviral vectors may be derived from nonhuman simian adenoviruses isolated from bonobos, such as PanAd l, PanAd2 or PanAd3. Examples of such vectors described herein can be found for example in WO2005/071093 and WO2010/086189.
• Adenoviral vectors may also be derived from adenoviruses isolated from gorillas as described in WO2013/52799, WO2013/52811 and WO2013/52832.
• Adenoviruses have a characteristic morphology with an icosahedral capsid comprising three major proteins, hexon (I I), penton base (I II) and a knobbed fiber (IV), along with a number of other minor proteins, VI, VI II, IX, Ilia and IVa2.
• the hexon accounts for the majority of the structural components of the capsid, which consists of 240 trimeric hexon capsomeres and 12 penton bases.
• the hexon has three conserved double barrels, while the top has three towers, each tower containing a loop from each subunit that forms most of the capsid.
• Penton is another adenoviral capsid protein that forms a pentameric base to which fiber attaches.
• the trimeric fiber protein protrudes from the penton base at each of the 12 vertices of the capsid and is a knobbed rod-like structure.
• the primary role of the fiber protein is the tethering of the viral capsid to the cell surface via the interaction of the knob region with a cellular receptor, and variations in the flexible shaft as well as knob regions of fiber are characteristic of the different serotypes (Nicklin et al Molecular Therapy 2005 12:384-393).
• Adenoviral vectors may be used to deliver desired RNA or protein sequences, for example heterologous sequences, for in vivo expression.
• a vector may include any genetic element including naked DNA, a phage, transposon, cosmid, episome, plasmid, or a virus.
• expression cassette or “minigene” is meant the combination of a selected heterologous gene (transgene) and the other regulatory elements necessary to drive translation, transcription and/or expression of the gene product in a host cell.
• an adenoviral vector is designed such that the expression cassette is located in a nucleic acid molecule which contains other adenoviral sequences in the region native to a selected adenoviral gene.
• the expression cassette may be inserted into an existing gene region to disrupt the function of that region, if desired.
• the expression cassette may be inserted into the site of a partially or fully deleted adenoviral gene.
• the expression cassette may be located in the site of a mutation, insertion or deletion which renders non-functional at least one gene of a genomic region selected from the group consisting of E1A, E1B, E2A, E2B, E3 and E4.
• the term "renders nonfunctional” means that a sufficient amount of the gene region is removed or otherwise disrupted, so that the gene region is no longer capable of producing functional products of gene expression.
• the entire gene region may be removed (and suitably replaced with the expression cassette).
• El genes of adenovirus are deleted and replaced with an expression cassette consisting of the promoter of choice, cDNA sequence of the gene of interest and a poly A signal, resulting in a replication defective recombinant virus.
• the transgene encoded by the adenoviral vector is a sequence encoding a product which is useful in biology and medicine, such as therapeutic or immunogenic proteins, RNA, enzymes, or catalytic RNAs.
• Desirable RNA molecules include tRNA, dsRNA, ribosomal RNA, catalytic RNAs, RNA aptamers, and antisense RNAs.
• a useful RNA sequence is a sequence which extinguishes expression of a targeted nucleic acid sequence in the treated animal.
• the mixture or composition as described herein is for use in prophylactic (thus immunogenic or preventive) or therapeutic treatment of a subject, such as a mammal or human subject, depending on the transgene encoded by the adenoviral vector.
• the transgene may encode a polypeptide or protein used for treatment, e.g., of genetic deficiencies, as a cancer therapeutic or vaccine, for induction of an immune response, and/or for prophylactic vaccine purposes.
• induction of an immune response refers to the ability of a protein, also known as an "antigen" or “immunogen", to induce a T cell and/or a humoral immune response to the protein.
• Immunogens expressed by the adenoviral vectors formulated as described herein and which are useful to immunize a human or non-human animal against other pathogens include, e.g., bacteria, fungi, parasitic microorganisms or multicellular parasites which infect human and non-human vertebrates, or from a cancer cell or tumor cell.
• immunogens may be selected from a variety of viral families.
• the immunogen is from a filovirus, for example Ebola (Zaire, Sudan, Reston, Budibugyo and Ivory Coast species) or Marburg.
• Ebola Zaaire, Sudan, Reston, Budibugyo and Ivory Coast species
• Such antigens may be derived from the viral glycoprotein (transmembrane and/or secreted form) and/or the viral nucleoprotein.
• immunogens may be selected from respiratory viruses such as respiratory syncytial virus (RSV) and other paramyxoviruses such as human metapneumovirus, hM PV and parainfluenza viruses (PIV).
• RSV respiratory syncytial virus
• PIV parainfluenza viruses
• Suitable antigens of RSV which are useful as immunogens to immunize a human or non-human animal can be selected from: the fusion protein (F), the attachment protein (G), the matrix protein (M2) and the nucleoprotein (N).
• F the fusion protein
• G attachment protein
• M2 matrix protein
• N nucleoprotein
• ChAdl55-RSV construct as disclosed in
• the immunogen may be from a retrovirus, for example a lentivirus such as the Human Immunodeficiency Virus (HIV).
• HIV Human Immunodeficiency Virus
• immunogens may be derived from HIV- 1 or HIV-2 sequences, such as for Gag, Pol, Nef, Env, and others. Such vectors are described, inter alia, in G B1510357.5 and WO2008/107370.
• the immunogen may be from Human Papilloma Virus (HPV).
• HPV Human Papilloma Virus
• immunogens may be derived from any HPV type and in particular from HPV types known to cause illness or disease, e.g. high risk H PV types causing urogenital cancers H PV16, HPV18 and the like.
• a transgene sequence may include a reporter sequence, which upon expression produces a detectable signal.
• reporter sequences include, without limitation, DNA sequences encoding ⁇ -lactamase, ⁇ -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (G FP), chloramphenicol acetyltransferase (CAT), luciferase, membrane bound proteins including, for example, CD2, CD4, CD8, the influenza hemagglutinin protein, and others well known in the art, to which high affinity antibodies directed thereto exist or can be produced by conventional means, and fusion proteins comprising a membrane bound protein appropriately fused to an antigen tag domain from, among others, hemagglutinin or Myc.
• DNA sequences encoding ⁇ -lactamase, ⁇ -galactosidase (LacZ), alkaline phosphatase, thymidine kinase, green fluorescent protein (G FP), chloramphenicol acetyltransferase (CAT),
• the expression cassette when associated with regulatory elements which drive their expression, provide signals detectable by conventional means, including enzymatic, radiographic, colorimetric, fluorescence or other spectrographic assays, fluorescent activating cell sorting assays and immunological assays, including enzyme linked immunosorbent assay (ELISA), radioimmunoassay (RIA) and immunohistochemistry.
• the expression cassette also may include conventional control elements which are operably linked to the transgene in a manner that permits its transcription, translation and/or expression in a cell transfected with the adenoviral vector.
• "operably linked" sequences include both expression control sequences that are contiguous with the gene of interest and expression control sequences that act in trans or at a distance to control the gene of interest.
• Expression control sequences include appropriate transcription initiation, termination, promoter and enhancer sequences; efficient RNA processing signals such as splicing and polyadenylation (poly A) signals including rabbit beta-globin polyA; sequences that stabilize cytoplasmic mRNA; sequences that enhance translation efficiency (e.g., Kozak consensus sequence); sequences that enhance protein stability; and when desired, sequences that enhance secretion of the encoded product.
• RNA processing signals such as splicing and polyadenylation (poly A) signals including rabbit beta-globin polyA
• sequences that enhance translation efficiency e.g., Kozak consensus sequence
• sequences that enhance protein stability e.g., Kozak consensus sequence
• sequences that enhance protein stability e.g., chimeric introns may be used.
• a “promoter” is a nucleotide sequence that permits binding of RNA polymerase and directs the transcription of a gene.
• a promoter is located in the 5' non-coding region of a gene, proximal to the transcriptional start site of the gene. Sequence elements within promoters that function in the initiation of transcription are often characterized by consensus nucleotide sequences. Examples of promoters include, but are not limited to, promoters from bacteria, yeast, plants, viruses, and mammals (including humans).
• a great number of expression control sequences, including promoters which are internal, native, constitutive, inducible and/or tissue-specific, are known in the art and may be utilized.
• Adenoviral vectors are generated by the modification of the wild type adenovirus to express heterologous genes (transgenes) and/or delete or inactivate undesirable adenoviral sequences.
• Adenoviral vectors may also have altered replication competency.
• the vector may be replication defective or have limited replication such that it has a reduced ability to replicate in non- complementing cells, compared to the wild type virus. This may be brought about by mutating the virus e.g. by deleting a gene involved in replication, for example deletion of the Ela, Elb, E3 or E4 gene.
• Such modifications are known to the skilled person and described in the art, e.g. by Roy et al., Human Gene Therapy 15:519-530, 2004; Colloca et al. (2012) 5c/. Transl. Med. 4: 1-9; Roy et al. (2004) Virol. 324: 361-372; or WO 03/000283.
• vectors are generated using techniques known to those of skill in the art. Such techniques include conventional cloning techniques of cDNA such as those described in texts, use of overlapping oligonucleotide sequences of the adenovirus genomes, polymerase chain reaction, and any suitable method which provides the desired nucleotide sequence. Particularly suitable methods include standard homologous recombination methods such as those provided in Colloca et al. (2012) 5c/. Transl. Med. 4:1-9; Roy et al. (2004) Virol. 324: 361-372; Roy et al. (2010) J. Gene Med. 13: 17-25; and WO2010/085984 or recombineering methods as described in Warming et al. Nuc. Acids Res.
• the adenoviral vectors can be produced on any suitable cell line in which the virus is capable of replication.
• complementing cell lines which provide the factors missing from the viral vector that result in its impaired replication characteristics (such as El) can be used.
• a cell line may be HeLa (ATCC Accession No. CCL 2), A549 (ATCC Accession No. CCL 185), HEK 293, KB (CCL 17), Detroit [e.g., Detroit 510, CCL 72) and WI-38 (CCL 75) cells, among others.
• HeLa ATCC Accession No. CCL 2
• A549 ATCC Accession No. CCL 185
• HEK 293, KB CCL 17
• Detroit e.g., Detroit 510, CCL 72
• WI-38 CCL 75
• Procell92 cell line is based on HEK 293 cells which express adenoviral El genes, transfected with the Tet repressor under control of the human phosphoglycerate kinase-1 (PGK) promoter, and the G418-resistance gene (Vitelli et al. PLOS One (2013) 8(e55435): l-9).
• Procell92.S is adapted for growth in suspension conditions and is also useful for producing adenoviral vectors expressing toxic proteins
• a mixture or composition as described herein may comprise one or more recombinant vectors capable of inducing an immune response, for example a humoral (e.g., antibody) and/or cell-mediated (e.g., a cytotoxic T cell) response, against a transgene product delivered by the vector following delivery to a mammal, suitably a human.
• a recombinant adenovirus may comprise (suitably in any of its gene deletions) a gene encoding a desired immunogen and may therefore be used in a vaccine.
• the recombinant adenoviruses can be used as prophylactic or therapeutic vaccines against any pathogen for which the antigen(s) crucial for induction of an immune response and able to limit the spread of the pathogen has been identified and for which the cDNA is available.
• the mixture and/or composition described herein are for use in
• the aqueous mixture and/or (freeze-dried) compositions described herein may be administered to a mammal, e.g. to a human subject.
• those mixtures or compositions comprising adenoviral vector encoding a transgene I.e. a recombinant adenoviral vector
• those mixtures or compositions comprising adenoviral vector encoding a transgene I.e. a recombinant adenoviral vector
• those mixtures or compositions comprising adenoviral vector encoding a transgene I.e. a recombinant adenoviral vector
• those mixtures or compositions comprising adenoviral vector encoding a transgene I.e. a recombinant adenoviral vector
• a transgene I.e. a recombinant adenoviral vector
• a mixture or composition of the invention may be formulated to contain other components, including, e.g., further immunogen(s), e.g. polypeptide antigen(s), and/or adjuvants.
• an adjuvant can be administered with a priming DNA vaccine encoding an antigen to enhance the antigen-specific immune response compared with the immune response generated upon priming with a DNA vaccine encoding the antigen only.
• an adjuvant can be administered with a polypeptide antigen which is administered in an administration regimen involving the adenoviral vectors of the invention.
• the mixture or composition as described herein is administered to a subject by intramuscular injection, intravaginal administration, intravenous injection, intraperitoneal injection, subcutaneous injection, epicutaneous administration, intradermal administration, nasal administration or oral administration.
• the therapeutic regimen involves co-administration of one or more adenoviral vectors and/or a further component
• these may be coformulated (I.e. in the same mixture or composition) or each formulated in different compositions.
• they are favourably administered co-locationally at or near the same site.
• the components can be administered (e.g. via an administration route selected from intramuscular, transdermal, intradermal, sub-cutaneous) to the same side or extremity ("co-lateral" administration) or to opposite sides or extremities ("contralateral” administration).
• Dosages of the viral vector will depend primarily on factors such as the condition being treated, the age, weight and health of the patient, and may thus vary among patients. For example, a
• therapeutically effective adult human or veterinary dosage of the viral vector generally contains lxlO 5 to lxlO 15 viral particles, such as from lxlO 8 to lxlO 12 (e.g., lxlO 8 , 5xl0 8 , lxlO 9 , 5xl0 9 , lxlO 10 , 2.5xl0 10 , 5xl0 10 , lxlO 11 5x1 ⁇ 11 , lxlO 12 particles).
• lxlO 8 , 5xl0 8 , lxlO 9 , 5xl0 9 , lxlO 10 , 2.5xl0 10 , 5xl0 10 , lxlO 11 5x1 ⁇ 11 , lxlO 12 particles e.g., lxlO 8 , 5xl0 8 , lxlO 9 , 5xl0 9 , lxlO 10 , 2.5xl0 10 , 5xl
• a viral vector can be administered at a dose that is typically from lxlO 5 to lxlO 10 plaque forming units (PFU), such as lxlO 5 PFU, 5x10 s PFU, 1x106 PFU, 5xl0 6 PFU lxlO 7 PFU, 5xl0 7 PFU, lxlO 8 PFU, 5xl0 8 PFU, lxlO 9 PFU, 5xl0 9 PFU, or lxlO 10 PFU. Dosages will vary depending upon the size of the animal and the route of administration.
• PFU plaque forming units
• a suitable human or veterinary dosage for about an 80 kg animal for intramuscular injection is in the range of about 1 x 10 9 to about 5 x 10 12 particles per mL, for a single site.
• multiple sites of administration may be used.
• a suitable human or veterinary dosage may be in the range of about 1 x 10 11 to about 1 x 10 15 particles for an oral formulation.
• the adenoviral vector can be quantified by Quantitative PCR Analysis (Q-PCR), for example with primers and probe designed on CMV promoter region using as standard curve serial dilution of plasmid DNA containing the vector genome with expression cassette including HCMV promoter.
• the copy number in the test sample is determined by the parallel line analysis method.
• Alternative methods for vector particle quantification can be analytical HPLC or spectrophotometric method based on A260 nm.
• An immunologically effective amount of a nucleic acid may suitably be between 1 ng and 100 mg.
• a suitable amount can be from 1 ⁇ g to 100 mg.
• An appropriate amount of the particular nucleic acid (e.g., vector) can readily be determined by those of skill in the art.
• Exemplary effective amounts of a nucleic acid component can be between 1 ng and 100 ⁇ g, such as between 1 ng and 1 ⁇ g (e.g., 100 ng-1 ⁇ g), or between 1 ⁇ g and 100 ⁇ g, such as 10 ng, 50 ng, 100 ng, 150 ng, 200 ng, 250 ng, 500 ng, 750 ng, or 1 ⁇ g.
• Effective amounts of a nucleic acid can also include from 1 ⁇ g to 500 ⁇ g, such as between 1 ⁇ g and 200 ⁇ g, such as between 10 and 100 ⁇ g, for example 1 ⁇ g, 2 ⁇ g, 5 ⁇ g, 10 ⁇ g, 20 ⁇ g, 50 ⁇ g, 75 ⁇ g, 100 ⁇ g, 150 ⁇ g, or 200 ⁇ g.
• an exemplary effective amount of a nucleic acid can be between 100 ⁇ g and 1 mg, such as from 100 ⁇ g to 500 ⁇ g, for example, 100 ⁇ g, 150 ⁇ g, 200 ⁇ g, 250 ⁇ g, 300 ⁇ 3 ⁇ 4 400 ⁇ g, 500 ⁇ 3 ⁇ 4 600 ⁇ 3 ⁇ 4 700 ⁇ 3 ⁇ 4 800 ⁇ 3 ⁇ 4 900 ⁇ g or 1 mg.
• a human dose will be contained in a volume of between 0.3 ml and 2 ml.
• the mixture and/or composition described herein can be formulated such that upon reconstitution of the dried composition a volume of, for example 0.3, 0.4, 0.5, 0.6, 1.0, 1.5 or 2.0 ml human dose per individual or combined immunogenic components is administered.
• telomere length a single dose that is administered hourly, daily, weekly or monthly, or yearly.
• mammals may receive one or two doses containing between about 10 ⁇ g to about 50 ⁇ g of plasmid in carrier. The amount or site of delivery is desirably selected based upon the identity and condition of the mammal.
• the therapeutic levels of, or level of immune response against, the protein encoded by the selected transgene can be monitored to determine the need, if any, for boosters. Following an assessment of CD8+ T cell response, or optionally, antibody titers, in the serum, optional booster immunizations may be desired.
• the adenoviral vector may be delivered in a single administration or in various combination regimens, e.g., in combination with a regimen or course of treatment involving other active ingredients or in a prime-boost regimen.
• “therapy” or “therapeutic” may relate to either or both of preventive and curative therapy.
• the aqueous mixture or dried composition may be contained in a glass vial, either siliconized or non- siliconized.
• the aqueous mixture or dried composition are provided in a non- siliconized vial.
• the aqueous mixture can be contained in a non-siliconized vial and freeze- dried when contained in that vial.
• the invention also provides a kit comprising two containers, of which a first container comprises the adenoviral composition as defined herein and a second container comprises the liquid as defined herein for reconstitution of the dried composition.
• the invention also provides a method for lyophilising or freeze-drying a liquid containing an adenoviral vector, such as the aqueous mixture as defined herein, to obtain a freeze-dried composition as defined herein, the method comprising an annealing step. Lyophilisation or freeze-drying cycle usually consists of three process phases.
• the invention also provides a method for freeze- drying a liquid containing an adenoviral vector, such as the aqueous mixture as described herein, whereby the freezing step of the freeze-drying cycle comprises an annealing step. Freezing and drying temperature and time will ultimately determine the moisture content of the freeze-dried composition.
• the moisture content of the freeze-dried composition is 1.4% (w/w) or higher, e.g. between 1.4 and 10% (w/w), between 1.4 and 8% (w/w), between 1.7 and 8% (w/w), between 1.9 and 8% (w/w), between 1.4 and 5% (w/w), between 1.7 and 5% (w/w), between 1.9 and 5% (w/w), between 1.4 and 3% (w/w), between 1.7 and 3% (w/w), or between 1.9 and 3% (w/w).
• the moisture content of the freeze-dried composition is 1.7% (w/w) or higher, 1.8% (w/w) or higher, or, 1.9% (w/w) or higher.
• glass transition temperature or “Tg” is the temperature at which an amorphous solid becomes soft upon heating or brittle upon cooling.
• Tg' refers to the glass transition temperature in the frozen state.
• collapse temperature or “Tc” refers to the temperature at which an amorphous material softens to the extent that it can no longer support its own structure.
• freeze-drying and “lyophilising”, and, “freeze-dried” and “lyophilised” are used
• annealing step refers to a method step in freeze-drying cycles of a composition, wherein during the freezing phase the product is maintained at a specified subfreezing temperature for a predetermined period of time. As is known to the skilled person, annealing will lead to Oswald ripening of the ice crystals and cryoconcentration of the amorphous matrix. Typically, the annealing temperature is (slightly) above Tg'. In one embodiment, annealing is executed at a temperature between (Tg' + 0.5°C) and (Tg' + 20°C), e.g.
• the annealing temperature should be between Tg' and the melting temperature (Tm) during annealing.
• annealing is done at a temperature between -4°C and -24°C, alternatively between -4°C and -20°C, alternatively between -4°C and -15°C, or alternatively between -8°C and -15°C, e.g. at -10°C +/-0.5°C.
• Annealing can be done during the freezing of the product, i.e. whilst the frozen sample is being formed, provided the product is frozen (solid state) and in a glassy state (below Tg').
• annealing is done post freezing of the product.
• the annealing temperature is about -10°C (e.g. -10°C +/- 1°C), more in particular where the aqueous mixture comprises sorbitol and trehalose in a ratio of sorbitol to trehalose between 4/14 and 4/16.5.
• the product is frozen (i.e. product temperature below Tg') prior to the annealing step.
• freezing is achieved by exposing the sample or aqueous mixture to a constant shelf temperature at a freezing temperature which is below Tg'.
• the product may be frozen by applying shelf-ramp freezing, i.e. gradually reducing the shelf temperature to a freezing temperature below Tg'.
• the freezing temperature is a temperature below Tg' minus 5°C, below Tg' minus 7.5°C, or below Tg' minus 10°C, such as at or below -50°C.
• the product temperature i.e. the temperature of the sample in the freeze-drier
• the freeze-drying cycle is started is between +2°C and +8°C.
• the temperature is reduced at a rate of at least 0.1°C/min, at least 0.2°C/min, at least 0.3°C/min or at least 0.5°C/min, and/or a rate of less than 10°C/min, 7.5°C/min, 5°C/min or less than 3°C/min.
• the temperature is reduced at a rate of 0.1 to 10°C/min, 0.1 to 5°C/min, 0.2 to 3°C/min, or 0.3 to l°C/min.
• the shelf temperature reached is maintained for about or at least 1 hour (or 60 minutes).
• the shelf temperature is increased to a temperature above Tg' to initiate the annealing step, such as to a temperature above Tg' plus 0.5°C, above Tg' plus 1°C, above Tg' plus 3°C, above Tg' plus 5°C, above Tg' plus 10°C or above Tg' plus 20°C.
• the temperature is kept below Tm during annealing.
• the temperature is raised at a rate of at least 0.1°C/min, at least 0.2°C/min, at least 0.3°C/min or at least 0.5°C/min, and/or a rate of less than 10°C/min, 7.5°C/min, 5°C/min or less than 3°C/min.
• the temperature is raised at a rate of 0.1 to 10°C/min, 0.1 to 5°C/min, 0.2 to 3°C/min, or 0.3 to l°C/min.
• the annealing temperature is maintained for at least two and/or up to four hours.
• the shelf temperature is reduced to a temperature below Tg' prior to initiating the drying under reduced pressure, such as to a temperature below Tg' minus 5°C, below Tg' minus 7.5°C, or below Tg' minus 10°C, such as at or below -50°C.
• the temperature is reduced at a rate of at least 0.1°C/min, at least 0.2°C/min, at least 0.3°C/min or at least 0.5°C/min, and/or a rate of less than 10°C/min, less than 7.5°C/min, less than 5°C/min or less than 3°C/min.
• the temperature is reduced at a rate of 0.1 to 10°C/min, 0.1 to 5°C/min, 0.2 to 3°C/min, or 0.3 to l°C/min.
• the shelf temperature reached is maintained for about or at least 1 hour (or 60 minutes). Drying under reduced pressure as contemplated in step b.ii. of the lyophilisation method described herein will typically be done in two phases, i.e. primary drying and secondary drying. In an
• step b.ii. of the method will include:
• Step b.ii. l. for primary drying at a temperature below Tc of the product
• Step b.ii.2. for secondary drying at a temperature above Tc of the product and below the Tg of the product.
• primary drying of the compositions described herein is done at -30°C +/-5°C
• secondary drying of the compositions described herein is done at 10°C +/- 5°C
• primary drying of the compositions described herein is done at -30°C +/-5°C
• secondary drying is done at 10°C +/- 5°C.
• primary drying conditions are applied for 24 hours or longer, between 24 and 40 hours, or, between 30 and 40 hours.
• primary drying is done at a pressure lower than 90 ⁇ bar and/or above 40 ⁇ bar. Primary drying conditions may be applied for up to 24 hours or longer.
• secondary drying temperature being achieved by raising the shelf temperature at a rate of 0.1°C/min, at least 0.2°C/min, at least 0.3°C/min or at least 0.5°C/min, and/or a rate of less than 3°C/min, less than 2°C/min, or less than l°C/min.
• secondary drying temperature is achieved by raising the shelf temperature at a rate of 0.1 to 3°C/min, 0.2 to 2°C/min, or 0.3 to l°C/min.
• the secondary drying temperature is at least - 10°C and/or below 30°C.
• the secondary drying temperature for the sorbitol containing compositions is 25°C +/- 5°C.
• secondary drying temperature is 10°C +/- 5°C.
• Secondary drying conditions may be applied for at least or for about three hours, at least about four hours, at least about five hours, or, at least, or for about six hours.
• Embodiment 1 A composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM
• Embodiment 2 A composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose and (vii) 23% (w/v) trehalose.
• Embodiment 3 A composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) 0.02% (w/v) TWEEN 80.
• an adenoviral vector (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 4 A composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 5 A lyophilised or freeze-dried composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose and (viii) 3.5% (w/v) sorbitol.
• Embodiment 6 A lyophilised or freeze-dried composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose and (vii) 23% (w/v) trehalose.
• Embodiment 7 A lyophilised or freeze-dried composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 8 A lyophilised or freeze-dried composition comprising (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 9 A composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose and (viii) 3.5% (w/v) sorbitol.
• Embodiment 10 A composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose and (vii) 23% (w/v) trehalose.
• Embodiment 11 A composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 12 A composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 13 A lyophilised or freeze-dried composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose and (viii) 3.5% (w/v) sorbitol.
• an adenoviral vector consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose and (viii) 3.5% (w/v) sorbitol.
• Embodiment 14 A lyophilised or freeze-dried composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose and (vii) 23% (w/v) trehalose.
• Embodiment 15 A lyophilised or freeze-dried composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) 0.02% (w/v) TWEEN 80.
• an adenoviral vector consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v)
• Embodiment 16 A lyophilised or freeze-dried composition consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose and (ix) 0.02% (w/v) TWEEN 80.
• an adenoviral vector consisting essentially of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 17 A composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) water for injection.
• an adenoviral vector consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) water for injection.
• Embodiment 18 A composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose and (ix) water for injection.
• Embodiment 19 A composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol, (ix) 0.02% (w/v) TWEEN 80 and (ix) water for injection.
• an adenoviral vector consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol, (
• Embodiment 20 A composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose, (ix) 0.02% (w/v) TWEEN 80 and (ix) water for injection.
• an adenoviral vector consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose, (ix) 0.02% (w/v) TWEEN 80 and (ix) water for injection.
• Embodiment 21 A lyophilised or freeze-dried composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose and (viii) 3.5% (w/v) sorbitol.
• an adenoviral vector consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose and (viii) 3.5% (w/v) sorbitol.
• Embodiment 22 A lyophilised or freeze-dried composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose and (vii) 23% (w/v) trehalose.
• Embodiment 23 A lyophilised or freeze-dried composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol and (ix) 0.02% (w/v) TWEEN 80.
• an adenoviral vector consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 18% (w/v) trehalose, (viii) 3.5% (w/v) sorbitol
• Embodiment 24 A lyophilised or freeze-dried composition consisting of (i) an adenoviral vector, (ii) 10 mM TRIS, (iii) 10 mM Histidine, (iv) 5 mM NaCI, (v) 1 mM MgCI 2 , (vi) 2% (w/v) sucrose, (vii) 23% (w/v) trehalose and (ix) 0.02% (w/v) TWEEN 80.
• Embodiment 25 The composition of any one of Embodiments 1 to 24 in which the adenoviral vector is a simian adenovector, such as a chimpanzee adenovector.
• an adenoviral vector selected from ChAd3, ChAd63, ChAd83, ChAdl55, ChAdl57, Pan 5, Pan 6, Pan 7 or Pan 9 yet more particularly an adenoviral vector is selected from ChAd3, ChAd63, ChAd83, ChAdl55, ChAd l57, and PanAd3 and still yet more particularly the adenoviral vector is ChAdl55.
• ChAdl55 vector used in the experiment encodes a respiratory syncytial viral protein (ChAdl55-RSV) and is described in PCT/EP2016/063297.
• the ChAdl55 particles were formulated in an aqueous mixture further comprising the excipients Tris 10 mM - Histidine 10 mM - MgCI2.6H20 1 mM - Tween 80 0.02% (w/v) - NaCI 5 mM - Trehalose or Sucrose 23% (w/v) - Sorbitol 2% (w/v). Sugar concentration was calculated to reach the maximum osmolality allowed for paediatric injection, i.e. 900 mOsm/kg. The concentration of the viral particles was l.lxlO 11 pU/ml.
• the shelf temperature was set at -52°C.
• the filled vials were loaded into the freeze dryer when the shelf temperature was at or lower than -45°C.
• the samples were then cooled at -52°C for a minimum of one hour.
• the chamber pressure was set at 80 ⁇ bar and the shelf temperature was raised from - 50°C to -25°C over three hours. Shelf temperature and chamber pressure were maintained for 24 hours.
• the shelf temperature was raised from - 25 °C to +10 °C over six hours, whilst the chamber pressure was reduced at 40 ⁇ bar. When the shelf temperature reached +10 °C, these conditions were maintained for six hours.
• the chamber was filled with dry nitrogen until a chamber pressure of 825 mbar was reached, and then the stoppers were fully inserted into the vials
• stoppering Once stoppering was completed, the chamber pressure was equilibrated to atmospheric pressure for unloading. The chamber temperature was maintained at +2 to +8°C until the vials were unloaded. The vials were then unloaded and over sealed with aluminium flip-off caps.
• the PicoGreen assay measures degradation of the viral particles.
• Quant-iTTM PicoGreen dsDNA reagent is an ultrasensitive fluorescent nucleic acid stain for quantifying double stranded DNA in solution.
• Infectivity HEXON of the adenoviral particles is measured by flow cytometric detection of cells stained for adenovirus hexon capsid protein.
• Adenovirus particle unit concentration is also measured using an Anion Exchange High Performance Liquid Chromatography (AEX-HPLC) system coupled with a fluorescence detector and using a commercial adenovirus standard as reference.
• the chromatographic systems used were Dionex Ultimate 3000 and Waters Acquity U PLC biocompatible (H-class).
• Compositions containing trehalose showed an increase of the glass transition temperature inducing a better stability at high temperature storage of the product. Also, infectivity increased (10-20%) in the trehalose formulations compared to sucrose formulations.
• the objective of the experiment was to evaluate several ranges of concentration of trehalose, sorbitol and NaCI to determine optimal conditions for the freeze-dried adenovirus candidate.
• the adenovirus used was ChAdl55-RSV.
• the protective effect of trehalose and sorbitol as observed in Example 1 was further evaluated together with the impact of NaCI.
• the ChAdl55-RSV particles were formulated in an aqueous mixture further comprising the excipients Tris 10 mM - Histidine 10 mM - MgCI2.6H20 ImM - Tween 80 0.02% (w/v) - NaCI (variable: 5, 25 or 45 mM) - Trehalose (14, 18.5 or 23% (v/w) )- Sorbitol (0, 2 or 4% (v/w) ).
• the concentration of the viral particles evaluated was l. lxlO 11 pU/ml.
• compositions were calculated to be reached after reconstitution of the freeze-dried composition with 0.625 ml of water for injection in non-siliconized type glass vials filled with 0.5 ⁇ 0.02 ml. Then the vials were partially sealed with a Helvoet FM460 bromobutyl stopper inserted in freeze drying position (partially inserted to allow water vapor to escape during the freeze drying cycle).
• sample 1 trehalose (T) 18.5% (v/w) - sorbitol (S) 0% (v/w) - NaCI (N) 25 mm
• freeze-drying cycle used comprised the following steps (as shown in Figure 4):
• the shelf temperature was set at -52°C.
• the filled vials were loaded into the freeze dryer when the shelf temperature was at or lower than -45°C.
• the samples were then cooled at -52°C for a minimum of one hour
• the chamber pressure was set at 80 ⁇ bar and the shelf temperature was raised from - 50°C to -30°C over three hours. Shelf temperature and chamber pressure were maintained for 24 hours
• the chamber was filled with dry nitrogen until a chamber pressure of 825 mbar was reached, and then the stoppers were fully inserted into the vials
• stoppering Once stoppering was completed, the chamber pressure was equilibrated to atmospheric pressure for unloading. The chamber temperature was maintained at +2 to +8°C until the vials were unloaded. The vials were then unloaded and over sealed with aluminium flip-off caps. All samples were analyzed at TO and after storage for one week at +4°C, +25°C (T1W25) or +30°C.
• Example 2 supported optimal ranges for trehalose (14 - 16%), Sorbitol (3 - 4%) and NaCI (5 mm). Further compositions were tested to complement the data of Example 2 using the same ChAdl55-RSV. For the compositions evaluated, the molarity of NaCI was fixed at 5 mm. Trehalose and sucrose content was varied as follows:
• freeze-drying cycle was evaluated in order to improve the stability of the cake appearance after storage (especially at +25°C (room temperature) in order to cover the time for reconstitution and administration to the patient after storage at cold temperature).
• Example 2 the impact of decreased Tg in the presence of sorbitol was determined to be directly correlated with the moisture content (residual humidity) measured in the cake after the freeze-drying step.
• the freeze-drying or lyophilisation cycle was further optimized as follows: lyo cycle (1): longer primary drying phase (+10H) was used in combination with a higher secondary drying temperature (at +25°C) (see Figure 13).
• the freeze-dried products were evaluated at TO and after stability of one week at +4°C (T1W4), +25°C (T1W25) and +30°C (T1W30).
• CCI D50 infectivity is a measure of adenovirus titer.
• the Cell Culture Infectious Dose 50 (CCID50) is an end-point dilution assay which quantifies the amount of virus required to produce a cytopathic effect in 50% of cells in a certain volume.
• the titer is expressed in a log 10 scale (Log CCI D50/ml). It is measured by inoculating the dilution series samples on indicator cells. After the incubation time (7 days at 37°C for adenovirus) the adenovirus hexon was immunostained and the results determined microscopically.
• Figure 18 shows the stability of adenovirus formulated with either 18% trehalose + 3.5% sorbitol or 23% trehalose at 4°C over 200 days, at a secondary drying temperature of 15°C.
• Figure 19 shows the stability of adenovirus formulated with either 18% trehalose + 3.5% sorbitol or 23% trehalose at 4°C over the course of 200 days, at a secondary drying temperature of 25°C.
• a concentration of 23% trehalose resulted in a relatively higher loss (approximately 45%) than a concentration of 18% trehalose + 3.5% sorbitol upon freeze-drying, with little subsequent loss at 4°C over time.
• 18% trehalose + 3.5% sorbitol resulted in an adenoviral vector loss of only approximately 30%.
• Example 5 Physical stability of lyophilised adenovirus
• Figure 20 shows the proportion of lyophilised samples that either remained intact (O), were cracked (+) or were fragmented (X) after a simulated transport of two hours via road transport and two hours via air transport.
• Formulation with 18% trehalose + 3.5% sorbitol resulted in lyophilised compositions that were less affected by vibrational stress than formulations with 23% trehalose.
• a secondary desiccation temperature of 15°C also resulted in lyophilised compositions that were less affected by vibration stress during transport.
• Formulation with 18% trehalose +3.5% sorbitol, with a secondary drying temperature of 15°C and the use of siliconized vials resulted in the best conditions for preserving the lyophilised cakes intact.
• Figure 21 shows the consistency of lyophilised samples that remained not powdery (O), slightly powdery (+) or powdery (X) after a simulated transport of two hours via road transport and two hours via air transport.
• Formulation with 18% trehalose + 3.5% sorbitol resulted in lyophilised compositions that were less affected by vibrational stress than formulations with 23% trehalose.
• a secondary desiccation temperature of 15°C also resulted in lyophilised compositions that were less affected by vibration stress during transport.
• a secondary drying temperature of 15°C resulted in less powder formation than a secondary drying temperature of 25°C.

Landscapes

• Health & Medical Sciences (AREA)
• Life Sciences & Earth Sciences (AREA)
• Chemical & Material Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Veterinary Medicine (AREA)
• Animal Behavior & Ethology (AREA)
• Medicinal Chemistry (AREA)
• Pharmacology & Pharmacy (AREA)
• Public Health (AREA)
• Engineering & Computer Science (AREA)
• Genetics & Genomics (AREA)
• Epidemiology (AREA)
• Organic Chemistry (AREA)
• Chemical Kinetics & Catalysis (AREA)
• General Chemical & Material Sciences (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Biotechnology (AREA)
• Molecular Biology (AREA)
• Biochemistry (AREA)
• Zoology (AREA)
• Wood Science & Technology (AREA)
• General Engineering & Computer Science (AREA)
• Biomedical Technology (AREA)
• Virology (AREA)
• Immunology (AREA)
• Oncology (AREA)
• Communicable Diseases (AREA)
• Physics & Mathematics (AREA)
• Dermatology (AREA)
• Biophysics (AREA)
• Oil, Petroleum & Natural Gas (AREA)
• Plant Pathology (AREA)
• Microbiology (AREA)
• Manufacturing & Machinery (AREA)
• Inorganic Chemistry (AREA)
• Tropical Medicine & Parasitology (AREA)
• Medicines Containing Material From Animals Or Micro-Organisms (AREA)
• Medicinal Preparation (AREA)
• Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)

Applications Claiming Priority (2)

Publications (1)

Family

ID=58462949

Family Applications (1)

Country Status (10)

Families Citing this family (6)

Family Cites Families (17)

• 2017
  • 2017-01-25 GB GBGB1701239.4A patent/GB201701239D0/en not_active Ceased
• 2018
  • 2018-01-25 JP JP2019539953A patent/JP7090089B2/ja active Active
  • 2018-01-25 MX MX2019008798A patent/MX2019008798A/es unknown
  • 2018-01-25 WO PCT/IB2018/050453 patent/WO2018138667A1/en unknown
  • 2018-01-25 CA CA3050629A patent/CA3050629A1/en active Pending
  • 2018-01-25 EP EP18703633.0A patent/EP3573598A1/en active Pending
  • 2018-01-25 CN CN201880021100.9A patent/CN110430867B/zh active Active
  • 2018-01-25 US US16/478,232 patent/US11590243B2/en active Active
  • 2018-01-25 BE BE2018/5040A patent/BE1025187B1/fr not_active IP Right Cessation
  • 2018-01-25 BR BR112019015245A patent/BR112019015245A2/pt active Search and Examination

Also Published As

Similar Documents

Legal Events